The development of computer tomography and image processing has made it possible to establish virtual 3D reconstruction and non-invasive dissection of fossil specimens. We used these methods to reconstruct a virtual 3D skull of a Pleistocene cheetah skull from the Tuozi cave, Tangshan, Nanjing, and virtually dissected it for anatomic studies, and measured the volumes of different parts of the endocranium. The endocranium of the cheetah skull has showed that its frontal sinus is beehive-tike, the frontal lobe of cerebra is relatively large but the temporal lobe is relatively small, its cerebral sulcus and gyrus are more complicated than those of the domestic cat, similar to those of the domestic dog, but simpler than those of giant panda, pig, cattle and horse. The technology of virtual 3D reconstruction and non-invasive dissection of fossil specimens can extend the morphological study from the exterior to the interior, and it can also help to study fragile specimens and virtually backup rare and precious specimens.